Large quantities of amylases are used industrially to carry out starch hydrolysis reactions, frequently referred to as enzymatic liquefaction and saccharification of starch. Such reactions are used to produce adhesives, sizings, maltodextrins, starch syrups of various compositions, maltose and dextrose. For industrial processes, it is particularly useful to have enzymes which are thermostable, which operate at selected pH ranges and which show resistance to heavy metal poisoning. Because of the commercial importance of amylases, there are great incentives to find low-cost sources for those enzymes having specific desired properties.
The amylases can be obtained from various microorganisms. It is known that these microorganisms contain genetic material which codes for the production of the enzymes by the organism. This genetic material is present in the form of DNA within the cell.
By techniques referred to as genetic engineering, it is possible to transfer a portion of the DNA from one organism to another. Attempts have been made to use these techniques to develop microorganisms which are amylase producers.
Two excellent reviews on the subject of genetic engineering are "DNA Cloning and the Analysis of Plasmid Structure and Function" by K. N. Timmis, S. N. Cohen and S. C. Cabello, Prog. Molec. subcell Biol., 6, 1978, pp. 1-58, and "Lamboid Phages that Simplify the Recovery of in vitro Recombinants" by Noreen E. Murray, W. J. Brammar and K. Murray, Molec. gen. Genet., 150, 1977, pp. 53-61. These reviews include many reports describing new, genetically modified microorganisms having valuable properties. Japanese patent publication No. SHO 52-76480 (published June 27, 1977, filed December 19, 1975 as SHO 50-150641) to Maruo, et al, discloses the manufacture of high-amylase producing strains of microorganisms by the in vivo techniques of mutagenesis, transduction and transformation to accumulate several genetic traits promoting amylase production in a Bacillus microorganism. These techniques, which do not comprise genetic engineering as herein defined, are limited to a single or a few closely related (genetically speaking) microorganisms. Also, these strains are not amenable to the gene amplification (e.g., by phage or plasmid) used in the instant invention for higher enzyme production.
In a recent article by Yuko Yoneda, Scott Graham and Frank E. Young, entitled "Cloning of a Foreign Gene Coding for alpha-Amylase in Bacillus subtilis", Biochemical and Biophysical Research Communications, 91, No. 4, pp. 1556-1564 (December 28, 1979), the authors describe the cloning of an alpha-amylase coding gene into a Bacillus subtilis (hereafter written B. subtilis) by bonding cleaved DNA of Bacillus amyloliquefaciensH with the DNA of the temperate phage phi 3T and subsequently transforming B. subtilis amylase-deficient cells. The authors do not show any evidence of amplification of the gene, with attendant massive production of the amylase enzyme, which is an important objective of the present invention.